Anti c myc

The agro-infiltrated leaf tissues were homogenized in 500 μl of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 2 mM Na₃VO₄, 25 mM β-glycerophosphate, 10 mM NaF, 0.05–0.1% Tween, 1 mM PMSF, pH 7.5). The homogenates were centrifuged at 14,000 × g for 20 min at 4 °C, then the supernatants were collected and the protein concentration was determined by the Bradford method^{64 (link)}. Aliquots of 50 μg of protein were separated on 8% acrylamide gels and were then electro-transferred to PVDF membranes using the Bio-Rad gel transfer apparatus in TBST buffer for 1 h. Membranes were blocked in a 3% BSA/TBST buffer solution at room temperature for 0.5 h, followed by incubation with the appropriate primary antibodies at 4 °C overnight [anti-FLAG, Abmart, cat # M20008 at 1:1000 dilution; anti-HA, Roche, cat # 12013819001 at 1:1000 dilution; anti-c-Myc, Millipore, cat # 05-724 at 1:1000 dilution). Following incubation, the membranes were washed three times with TBST and incubated with an HRP-conjugated goat, anti-mouse secondary antibody (Abmart, lot # M21001 at 1:1000 dilution). The blots were washed again three times with TBST buffer and the immunoreactive bands were detected using an enhanced chemiluminescence method.

The chromatin immunoprecipitation (ChIP) assay was performed using anti- c-myc and a ChIP assay kit (Millipore, USA) according to the manufacturer’s instructions. Anti-IgG antibody was used as the negative control. Briefly, HOS-MNNG and ZOSM cells were fixed, harvested, and sonicated, and the samples immunoprecipitated with the respective antibodies. The bound DNA fragments were subjected to qPCR using four primer sets to amplify the c-myc-binding region(s) of the SPTLC1 promoter. Primers used to amplify specific genomic regions are listed in Additional file 1: Table S1.

Western blotting analysis was performed according to standard methods as previously described.^{12 (link)} The following primary antibodies were used: anti-Sam68 (sc-333, dilution, 1:500; Santa Cruz Biotechnology, Delaware Ave Santa Cruz, CA), anti-beta-catenin, anticyclin D1, anti-p21^cip1, anti-p27^KIP1, anti-p-Rb, antitotal-Rb, anti-p-AKT, antitotal-AKT, anti-c-Myc (1:1000, Millipore, Billerica, MA), anti-P-84, anti-LEF-1 (1:500, Abcam, Cambridge, MA), anti-α-tubulin, anti-TCF-4, anti-LEF1, and anti-GAPDH (1:1000, Sigma, Saint Louis, MO). Nuclear extracts were prepared using the Nuclear Extraction Kit (Active Motif), according to the manufacturer's instructions.

HUVEC and ISO-HAS-B cells were cultured on gelatin coated coverslips and were either left untreated or transfected for ectopic expression of FoxO1 mutants. Cells were fixed for 10 min with 4% paraformaldehyde, washed with PBS and then permeabilized with 0.5% Triton-100 × -PBS for 10 min. Subsequently cells were blocked with 5% FCS 0.01% Tween-20-PBS for 30 min followed by incubation with anti-aPKC (1:100; Beckton Dickison), anti-VE-cadherin (1:100; R&D Systems); anti-c-Myc (1:100; Millipore); and anti-FLAG-M2 (1:400; Sigma) overnight at 4°C. Coverslips were then washed and incubated with the appropriate secondary antibodies (Alexafluor fluorescently conjugated; 1:400; Invitrogen) and Hoescht 33342 or DAPI, and mounted with Vectashield.

FSEC at confluence were washed three times with cold PBS 1x supplemented with 2 mM sodium orthovanadate, and then lysed in ice with Complete Tablet buffer (Roche) supplemented with 2 mM sodium orthovanadate and 50 μM MG132 (Sigma-Aldrich). Thirty-five μg of proteins from each lysate were loaded on 15 or 5 % SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (PVDF, GE Healthcare Europe GmbH, Milan, Italy). They were incubated overnight at 4 °C with specific primary antibody: anti-PAX8 (1:500, Abnova, DBA ITALIA S.r.l., Milan, Italy), anti-VDR (1:200, Santa-Cruz), anti-p53 (1:500, Santa-Cruz), anti-cMyc (1:200, Millipore S.p.A., Milan, Italy), anti-Ki67 (1:500, Santa-Cruz), and anti-pan-Ras (1:500, Santa-Cruz). Protein expression was normalized and verified through ß-actin detection (1:5000; Sigma, Milan, Italy).